Plasma membrane integrity is of critical importance for cell homeostasis and function. Physical, chemical or metabolic disruption of the plasma membrane leads to a repair-or-die emergency in the cell. An efficient plasma membrane repair mechanism is essential for life because disruption of this process due to genetic mutations can result in a number of diseases including muscular dystrophy and associated cardiomyopathy. Previous studies conducted by ourselves and others demonstrate that several proteins, including dysferlin and MG53, mediate the membrane repair response in cardiomyocytes. However, the molecular mechanisms underlying this important physiological process has not been fully defined. Our preliminary data found that anoctamin 5 (Ano5) plays an essential role in membrane repair in myocytes. Ano5 belongs to the anoctamin protein family that includes at least ten proteins all possessing eight transmembrane domains with proved or putative calcium-activated chloride channel (CaCC) functions. Mutations in the ANO5 gene (encoding Ano5) lead to muscular dystrophies in human patients. However, there is little known about the molecular and cellular functions of Ano5 in cardiomyocytes and the molecular mechanisms underlying Ano5-mediated membrane repair remain poorly understood. The long-term goal of this research proposal is to understand the molecular and cellular mechanisms for Ano5 in heart physiology and disease. In pilot studies, we found that Ano5 is primarily localized on the endoplasmic/sarcoplasmic reticulum (ER/SR) and RNAi-silencing of Ano5 shows defective membrane repair in myocytes. This shows a new biological function of Ano5 in the cellular physiology of muscle cells. In this project, we will focus on testing the hypothesis that Ano5 is involved in the calcium-activated chloride channel (CaCC) activity through oligomerization on the sarcoplasmic reticulum (SR), and that Ano5 plays an essential role in plasma membrane repair of cardiomyocytes by promoting vesicle generation upon membrane damage. Our planned experiments will significantly advance understanding of cardiomyocyte membrane repair mechanisms by manipulating expression of Ano5 and the use of live cell imaging, biochemical markers, ex vivo and in vivo animal model studies. These data will begin to define potential therapeutic targets for the regulation of membrane repair, thereby treating the diseases associated with abnormal membrane stability. Disrupted plasma membrane integrity underlies a number of diseases including cardiomyopathy. Our project is designed to understand the molecular and cellular functions of Ano5 in muscle physiology and disease. These studies will aid in defining therapeutic targets for the treatment of heart diseases associated with compromised plasma membrane integrity through the regulation of Ano5-mediated membrane repair capacity.